Glycosylation method

ABSTRACT

The invention relates to microbial host cells engineered to produce glycoconjugate vaccines by stable integration of an acceptor protein and an oligosaccharyltransferase into the host&#39;s genome, wherein expression of the oligosaccharyltransferase is regulated.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/655,128, filed Jun. 24, 2015, which is the U.S. National Stage of International Application No. PCT/GB2014/050133, filed Jan. 17, 2014, which was published in English under PCT Article 21(2), which in turn claims the benefit of United Kingdom Application Nos. 1300956.8, filed Jan. 18, 2013, 1301023.6, filed Jan. 21, 2013, and 1309068.3, filed May 20, 2013.

FIELD OF THE INVENTION

The invention relates to microbial host cells engineered to produce glycoconjugate vaccines by stable integration of an oligosaccharyltransferase and optionally a carrier protein into the host's genome, wherein expression of the oligosaccharyltransferase is regulated.

BACKGROUND OF THE INVENTION

Bioavailability and bioactivity of small molecules, peptides, proteins or nucleic acids can be altered by glycosylation. Glycosylation is the transfer of an activated sugar moiety from a donor to an acceptor molecule and is catalysed by glycosyltransferases. Glycosylation of proteins occurs either co- or post-translationally by which a sugar moiety is attached to a nitrogen of asparagine or arginine side-chains forming N-linked glycans, or to the hydroxy oxygen of serine, threonine, tyrosine, lysine, or proline side-chains forming O-linked glycans, or less common forming C-linked glycans where the sugar is added to carbon on a tryptophan side-chain.

N-linked glycosylation is the most common post-translational modification and is carried out in the endoplasmic reticulum of eukaryotic cells. N-linked glycosylation can be of two main types; high mannose oligosaccharides which have two N-acetylglucosamines and complex oligosaccharides which include other types of sugar groups. The peptide motif contained in glycosylated polypeptides is Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline. This is catalysed by the enzyme oligosaccharyltransferase [OT]; see Yan & Lennarz J. Bioi. Chem., Vol. 280 (5), 3121-3124 (2005). OT catalyses the transfer of an oligosaccharyl moiety (Glc3Man9GlcNAc2) from the dolichol-linked pyrophosphate donor to the side chain of an Asn. A pentasaccharide core is common to all N-linked oligosaccharides and serves as the foundation for a wide variety of N-linked oligosaccharides. O-linked glycosylation is less common. Serine or threonine residues are linked via their side chain oxygen to sugars by a glycosidic bond. Usually N-acetylglucosamine is attached in this way to intracellular proteins.

Most bacterial glycoproteins are associated with the bacterial cell surface or are secreted, suggesting a role for glycoproteins in the interaction with the host's immune system. Studies on the gram negative pathogenic bacterium Campylobacter jejuni identified a gene cluster involved in the synthesis of lipo-oligosaccharides and N-linked glycoproteins. The protein glycosylation locus, a cluster of 12 genes comprising pglA-pglG, is involved in the glycosylation of over 30 glycoproteins. Interestingly, these genes can be used to modify lipopolysaccharide structures in Escherichia coli providing a genetic tool to express heterologous recombinant glycoproteins. Studies performed with C. jejuni carrying mutations in the pglB and pglE genes showed impaired colonisation abilities in mice.

Polysaccharide antigens interact directly with B cells and induce antibody synthesis in the absence of T-cells. This T-cell independent immune response is limited as antibody production is low and is not normally boosted by re-immunisation. The antibody isotypes are dominated by IgM and IgG2, which are short lived and are generally of low affinity for a specific antigen. The ability to enhance the immunogenicity of polysaccharide antigens can be achieved by conjugation of the polysaccharide to a protein carrier. Glycoconjugate vaccines for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are currently licensed for human use and are produced by linking the capsule (or other bacterial glycan-based structure such as lipooligosaccharide) from these bacteria to a protein toxoid. Whilst these vaccines provide a good level of immunity they are expensive and difficult to produce, requiring the purification of the glycan from the pathogenic organisms and chemical linkage to the carrier protein. The use of organic systems represents a more rapid and economical method for the production of glycoconjugates.

The production of glyconjugates in an E. coli expression system requires the co-expression of three genes [“tri-plasmid”]: an acceptor protein, a polysaccharide biosynthetic locus and, for the coupling reaction, an oligosaccharyltransferase enzyme. Optimisation of co-expression in just one host is a lengthy process as it requires a tailored choice of the plasmids and optimised growth and expression conditions which are dependent on several factors. Additionally, the yield of glycoconjugates using such expression systems are often very low, which makes it commercial not viable.

The tri-plasmid system has a number of disadvantages. Compatible origins of plasmid replication need to be combined, a process that can take several months and restricts the choice of available plasmids. The use of three plasmids represents a considerable metabolic burden on the host bacterial strain and often the contents of one of the plasmids fail to express. Three different antibiotic selection markers for each of the plasmids need to be combined. This represents another burden on the host strain and clashes often result in sub-cloning of antibiotic resistance genes to find combinations that match. Additionally, some antibiotics resistance combinations are legally forbidden in some strains. Some glycosyltransferases [e.g. PgIB enzyme] responsible for linking the glycan to the protein has multiple transmembrane domains and is toxic to bacterial cells. Once expressed it has been found to reduce the growth of the host bacterial strain considerably. This significantly inhibits glycoconjugate yield. Heterologous expression of the polysaccharide coding genes and the resulting enzymes that are generated can result in high levels of toxicity to the host. Therefore, with some glycans it is impossible to find a tri-plasmid combination that allows for glycoconjugate production.

An example of the tri-plasmid system is disclosed in WO2009/104074 which describes the optimised production of conjugates comprising a protein carrier linked to an antigenic polysaccharide in an E. coli tri-plasmid expression system. The yield using this system is increased significantly by growing cells carrying three genes on two plasmids in a bioreactor under carefully monitored growth condition.

The present disclosure relates to a method providing the stable integration of genes into a bacterial genome for the production of glycoconjugates controlled by constitutive and regulated promoter elements which results in high yields of glycoconjugate. The application discloses also a method for the genetic manipulation of transposons facilitating easy and efficient preparation of the transposable element which allows integration of genes into the genome in one single step and additionally comprising also a tool which permits easy removal of the resistance marker once the gene of interest has integrated into the host genome.

Statements of the Invention

According to an aspect of the invention there is provided an isolated nucleic acid molecule comprising a transcription cassette adapted for expression in a bacterial host cell said cassette comprising: a nucleotide sequence encoding a glycosyltransferase enzyme wherein the expression of said enzyme is regulatable and a nucleotide sequence encoding an acceptor polypeptide for an oligosaccharide or monosaccharide wherein the acceptor polypeptide includes an amino acid sequence motif which is a substrate for the glycosyltransferase enzyme.

In a preferred embodiment of the invention the transcription cassette further comprises a regulatable promoter functionally linked to at least the nucleotide sequence encoding said glycosyltransferase enzyme.

In a preferred embodiment of the invention said promoter includes an inducible nucleotide element conferring regulatable expression in response to an inducer.

In a preferred embodiment of the invention said inducer is IPTG.

In an alternative embodiment of the invention said promoter includes a repressible nucleotide element conferring regulatable expression in response to a repressor.

In a preferred embodiment of the invention said repressor is the LacIq repressor.

Bacterial expression systems that utilize inducers and repressors of gene expression are well known in the art and include modifications that are well established which enhance induction or repression of gene expression. For example is laclq carries a mutation in the promoter region of the lacI gene that results in increased transcription and higher levels of Lac repressor within the cells. Moreover, the Ptac, a strong hybrid promoter composed of the −35 region of the trp promoter and the −10 region of the lacUV5 promoter/operator and is strongly inducible.

In a preferred embodiment of the invention said glycosyltransferase is selected from the group consisting of: an oligosaccharyltransferase, a glucosyltransferase, a mannosyltransferase or a glucuronosyltransferase.

In a preferred embodiment of the invention said glycosyltransferase is an oligosaccharyltransferase,

In a preferred embodiment of the invention said nucleic acid molecule encodes an oligosaccharyltransferase selected from the group consisting of:

-   -   i) a nucleic acid molecule comprising a nucleotide sequence as         set forth in SEQ ID NO 1 or SEQ ID NO: 22;     -   ii) a nucleic acid molecule the complimentary strand of which         hybridizes under stringent hybridization to SEQ ID NO: 1 or SEQ         ID NO: 22 and encodes an oligosaccharyltransferase that         recognizes the amino acid motif: D/E-X-N-X-S/T, wherein X is any         amino acid except proline;     -   iii) a nucleic acid molecule comprising a nucleotide sequence         that is degenerate with respect to the nucleotide sequence in         SEQ ID NO: 1 and which encodes a polypeptide comprising an amino         acid sequence as represented in SEQ ID NO:2;     -   iv) a nucleic acid molecule comprising a nucleotide sequence         that is degenerate with respect to the nucleotide sequence in         SEQ ID NO: 22 and which encodes a polypeptide comprising an         amino acid sequence as represented in SEQ ID NO: 23;     -   v) a variant sequence that varies from the amino acid sequences         set forth in SEQ ID NO: 2 or 23 and which has         oligosaccharyltransferase activity.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used.

Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_(m) is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand.

The following is an exemplary set of hybridization conditions and is not limiting.

Very High Stringency (Allows Sequences that Share at Least 90% Identity to Hybridize)

-   -   i) Hybridization: 5×SSC at 65° C. for 16 hours     -   ii) Wash twice: 2×SSC at room temperature (RT) for 15 minutes         each     -   iii) Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity to Hybridize)

-   -   i) Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours     -   ii) Wash twice: 2×SSC at RT for 5-20 minutes each     -   iii) Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity to Hybridize)

-   -   i) Hybridization: 6×SSC at RT to 55° C. for 16-20 hours     -   ii) Wash at least twice:2×-3×SSC at RT to 55° C. for 20-30         minutes each.

In a preferred embodiment of the invention said acceptor polypeptide includes the amino acid motif: Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline.

In an alternative embodiment of the invention said acceptor polypeptide includes the amino acid motif: D/E-X-N-X-S/T, wherein X is any amino acid except proline.

In an alternative preferred embodiment of the invention said acceptor polypeptide including the amino acid motif D/E-X-N-X-S/T, wherein X is any amino acid except proline is selected from the group consisting of: DVNVT (SEQ ID NO: 24), EVNAT (SEQ ID NO: 25), DQNAT (SEQ ID NO: 26), DNNNT (SEQ ID NO: 27), DNNNS (SEQ ID NO: 28), DQNRT (SEQ ID NO: 29), ENNFT (SEQ ID NO: 30), DSNST (SEQ ID NO: 31), DQNIS (SEQ ID NO: 32), DQNVS (SEQ ID NO: 33), DNNVS (SEQ ID NO: 34), DYNVS (SEQ ID NO: 35), DFNVS (SEQ ID NO: 36), DFNAS (SEQ ID NO: 37), DFNSS (SEQ ID NO: 38), DVNAT (SEQ ID NO: 39), DFNVT (SEQ ID NO: 40) or DVNAS (SEQ ID NO: 41).

In a further preferred embodiment of the invention said transcription cassette comprises a nucleotide sequence that encodes one or more polysaccharide biosynthetic genes.

In a preferred embodiment of the invention said one or more biosynthetic genes are selected from the group consisting of the capsular polysaccharide or O antigen coding regions.

In a preferred embodiment of the invention said transcription cassette is adapted by the provision of one or more inverted repeat nucleotide sequences that are transposase recognition sequences.

In a preferred embodiment of the invention said recognition sequence comprises or consists essentially of the nucleotide sequences:

(SEQ ID NO:7) 5′-CTGTCTCTTGATCAGATCT-3′ at the I end; and

(SEQ ID NO: 8) 5′-ACTTGTGTATAAGAGTCAG-3′,

at the O end of the miniTn5 transposon.

In a preferred embodiment of the invention said transcription cassette further comprises a nucleotide sequence encoding a bacterial resistance marker gene.

In a preferred embodiment of the invention said bacterial resistance gene confers resistance to an antibiotic.

In a preferred of the invention said antibiotic is selected from the group consisting of: kanamycin, chloramphenicol, ampicillin, rifampicin, tetracycline, carbenecillin or Zeocin® [phleomycin: cas code 1006-33-0].

In a preferred embodiment of the invention said antibiotic is Zeocin®.

A skilled artisan is familiar with the use of different antibacterial markers. There are several classes of antibiotics targeting either the bacterial cell wall, cell membrane, interfere with essential bacterial enzymes, or target protein synthesis. Further, antibacterial antibiotics can target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria.

In a preferred embodiment of the invention said bacterial resistance marker gene is adapted by the provision of 5′ and 3′ flanking nucleotide sequences that include recombination sites.

In a preferred embodiment of the invention said recombination sites comprise or consist essentially of the nucleotide sequence:

5′ end loxP sequence: (SEQ ID NO: 9) 5′-ATAACTTCGTATAATGTAT GCTATACGAA GTAG-3′.

3′ end loxP sequence: (SEQ ID NO:10) 5′-ATAAC TTCGTATAATGTATGCTATA CGAAGTTAT-3′for the

CRE LOX system or

(SEQ ID NO: 11) 5′-GAAGT TCCTATTCCG AAGTTCCTAT TCTCTAGAAAGTATAGGAAC TTC-3′ and

(SEQ ID NO:12) 5′-GAAGTACCTATTC CGAAGTTCCT ATTCTCTAGA AAGTATAGGA ACTTC-3′ for the FRT FLP system.

According to a further aspect of the invention there is provided a vector comprising a transcription cassette according to the invention.

In a preferred embodiment of the invention said vector is a plasmid.

In an alternative preferred embodiment of the invention said vector is a transposon.

In a preferred embodiment of the invention said transposon is selected from the group consisting of: Tn5, Tn10, Himar1 and other mariner elements, Tn7, Tn917, Tn916.

In a preferred embodiment of the invention said transposon is Tn5.

According to a further aspect of the invention there is provided a bacterial cell genetically modified with a transcription cassette or vector according to the invention.

In a preferred embodiment of the invention said bacterial cell is a pathogenic Gram-positive bacterial cell.

In a preferred embodiment of the invention said bacterial cell is a pathogenic Gram-negative bacterial cell.

In a preferred embodiment of the invention said bacterial cell is a human pathogen.

In a preferred embodiment of the invention said human pathogen is selected from the group: Neisseria, Moraxella, Escherichia, Salmonella, Shigella, Pseudomonas, Helicobacter, Legionella, Haemophilus, Klebsiella, Enterobacter, Cronobacter and Serratia.

In a preferred embodiment of the invention said bacterial cell is a non-human pathogen.

In a preferred embodiment of the invention said non-human pathogen is selected from group: Mannheimia spp., Actinobacillus spp. e.g Actinobacillus pleuropneumoniae, Pasteurella spp., Haemophilus spp. or Edwardsiella spp.

In a preferred embodiment of the invention said bacterial cell is a zoonotic bacterial species.

In a preferred embodiment of the invention said zoonotic bacterial species is selected from the group: Brucella spp., Campylobacter spp., Vibrio spp., Yersina spp. and Salmonella spp.

According to a further aspect of the invention there is providing a bacterial cell culture comprising a genetically modified bacterial cell according to the invention.

According to an aspect of the invention there is provided a transcription cassette or vector according to the invention for use in the production of one or more glycoconjugates.

According to a further aspect of the invention there is provided a process for the production of one or more glycoconjugates comprising:

-   -   i) providing a bacterial cell culture according to the         invention;     -   ii) providing cell culture conditions; and     -   iii) isolating one or more glyconjugates from the bacterial cell         or cell culture medium.

According to a further aspect of the invention there is provided a cell culture vessel comprising a bacterial cell culture according to the invention.

In a preferred embodiment of the invention said cell culture vessel is a fermentor.

Bacterial cultures used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, bacteria are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The products produced can be isolated from the bacteria as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

An overview of known cultivation methods can be found in the textbookBioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of the bacterial strains in question. Descriptions of culture media for various bacteria can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).

As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassiumhydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

The fermentation media used according to the invention for culturing bacteria usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C., preferably at from 25° C. to 40° C., and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20° C. to 45° C. and preferably 25° C. to 40° C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.

The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.

However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the fatty acids present therein.

According to a further aspect of the invention there is provided a method for the stable integration of a transposable element into a bacterial genome comprising:

-   -   i) forming a cell culture preparation comprising a bacterial         cell and a transposon according to the invention;     -   ii) incubating the preparation to allow stable integration of         the transposon;     -   iii) selecting bacterial cells that have stably integrated the         transposon using culture conditions that select for bacterial         cells that are stable integrants; and     -   iv) cloning bacterial cells that have stably integrated the         transposon.

In a preferred method of the invention said selection is an antibiotic selection using a bacterial resistance marker gene.

In a preferred method of the invention the cloned bacterial cells are contacted with a vector comprising a nucleotide sequence that encodes a recombinase that deletes all or part of said bacterial resistance marker gene.

In a preferred embodiment of the invention said recombinase is specific for recombination sites comprising or consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 13 or 14.

According to a further aspect of the invention there is provided a process for the identification of novel glycoconjugates comprising:

-   -   i) forming a cell culture preparation comprising a bacterial         cell and a transposon according to the invention;     -   ii) incubating the preparation to allow stable integration of         the transposon;     -   iii) selecting bacterial cells that have stably integrated the         transposon using culture conditions that select for bacterial         cells that are stable integrants;     -   iv) cloning bacterial cells that have stably integrated the         transposon;     -   v) isolating glycoconjugates from the cloned bacterial cells or         cell culture medium; and     -   vi) analysing the monosaccharide or polysaccharide content of         said isolated glycoconjugate.

According to a further aspect of the invention there is provided a glycoconjugate formed by the process according to the invention.

According to a further aspect of the invention there is provided a method for the delivery of a transposable element to a bacterial cell comprising:

-   -   i) providing a vector adapted for high copy number replication         in a bacterial cell comprising: a nucleotide sequence of an         expression cassette according to the invention, a nucleotide         sequence encoding an antibiotic resistance gene cassette wherein         the antibiotic resistance gene cassette is flanked by         recombination sequences recognized by a recombinase and wherein         the cassettes are flanked by restriction sites for excision and         delivery into a transposon delivery vector;     -   ii) providing a transposon delivery vector comprising         restriction sites flanked by inverted repeats for delivery of a         transposable element to a bacterial cell;     -   iii) sub-cloning the vector in (i) into the delivery vector         in (ii) to provide a modified delivery vector;     -   iv) transferring the modified delivery vector in (iii) to a         bacterial cell and selecting stably integrated bacterial cells         by antibiotic resistance;     -   v) cloning stably integrated bacterial cells; and optionally     -   vi) transforming the stably integrated bacterial cells with a         vector comprising a nucleotide sequence encoding a recombinase         that excises the antibiotic resistance genes from the genome of         the stably integrated bacterial cells.

Delivery vectors for the transposable elements such as TN5 transposons are known in the art and have significant limitations. For example, the pUT delivery vector includes a R6K origin of replication which limits transformation to E. coli strains that are A pir positive. The development of a high copy number intermediate vector considerably expands the type of bacterial host cell and significantly simplifies the modification of bacterial cells using transposable element.

According to a further aspect of the invention there is provided a kit comprising: a vector adapted for high copy number replication in a bacterial cell wherein the vector comprises: a nucleotide sequence encoding an antibiotic resistance gene cassette and a multiple cloning site for the insertion of an expression cassette according to the invention wherein the antibiotic gene cassette is flanked by recombination sequences recognized by a recombinase and wherein the multiple cloning sites are flanked by restriction sites enabling the excision of the expression cassette.

In a preferred embodiment of the invention said kit further comprises a transposon delivery vector comprising restriction sites flanked with transposase recognition nucleotide sequences.

In a further preferred embodiment of the invention said kit further comprises a recombinase, for example a flippase.

In a preferred embodiment of the invention the kit further includes a bacterial strain or strains compatible with the transposon.

In a preferred embodiment of the invention said kit optionally includes a nucleic acid molecule encoding a glycosyltransferase and a nucleic acid molecule that encodes one or more polysaccharide biosynthetic genes.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures;

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The tri-plasmid system currently used to generate a glycoconjugate vaccine in E. coli. C, cytoplasm; IM, inner membrane; P, periplasm; OM, Outer Membrane;

FIG. 2: Chromosomal insertion CjPglB generates superior glycoconjugate yield compared to the standard three plasmid system; AcrA purified from E. coli CLM24 carrying chromosomal PglB tested by western blot (lanes 2/4/6) compared to plasmid borne PglB (lanes 1/3/5);

FIG. 3A PglB nucleotide sequence (SEQ ID NO: 1);

FIG. 3B the full length PgIB amino acid sequence (SEQ ID NO: 2);

FIG. 4 is the nucleotide sequence encoding for the CRE LOX system recombinase (SEQ ID 13);

FIG. 5 is the nucleotide sequence encoding the FLP FRT recombinase (SEQ ID 14);

FIG. 6 Glycosylation of AcrA with S. pneumoniae capsular polysaccharide. Anti-HIS western blot. Lane 1, AcrA only control; 2 E. coli W3110 carrying AcrA and PglB on the chromosome due to an insertion of pEFNOV19: Coding for an IPTG inducible copy of CjPglB and constitutively expressed AcrA from a Tetracycline resistance cassette promoter on a pUTminiTn5km2 backbone. Arrows indicate shift in the size of AcrA only when all the glycosylating components are together; and

FIG. 7 Demonstration of acceptor protein glycosylation. Western blot using anti-glycan (B), anti-His (C) and both antibodies combined (A). AcrA glycosylated with C. jejuni heptasaccharide within E. coli W3110 cells carrying unmarked pglB insertion.

FIG. 8 In vitro glycosylation of peptide using pglB expressed in A. pleuropneumoniae (App). Reduced mobility of peptide is seen only in test lanes 2 and 9 where membranes from A. pleuropneumoniae coding for PglB are used. Lane 3 is a positive control reaction carrying E. coli membranes expressing PglB and Lanes 1, 4, 5, 6, 7, 8 and 10 are negative controls demonstrating that peptide shift only occurs when pglB is expressed in A. pleuropneumoniae or E. coli and combined with the correct glycan.

FIG. 9 is the nucleotide sequence of Campylobacter sputorum PglB (SEQ ID NO: 22); and

FIG. 10 Campylobacter sputorum protein sequence of PglB (SEQ ID NO: 23).

Materials and Methods

Construction of pJAN25

The gene coding for C. jejuni PglB was amplified by PCR with the pTac promoter and Lacl repressor from plasmid pEXT20 as a template using Pfx Polymerase with (SEQ ID 15: 5′-TTTTGCGGCCGCTTCTACGTGTTCCGCTTCC-3′) as forward primer and (SEQ ID 16: 5′-TTTTGCGGCCGCATTGCGTTGCGCTCACTGC-3′) reverse primer using the following cycling conditions, 94° C./2 minutes followed by 35 cycles of 94° C. for 30 seconds, 56° C. for 30 seconds and 68° C. for 4 minutes. The PCR product was then cloned in pJET2.0 plasmid (Thermo Scientific U.K.) according to the manufacturer's instructions and named pOST9. The plasmid was maintained in into E. coli DH5a (Stratagene U.K.) The vector pOST9 cut with the restriction enzyme NotI (New England Biolabs U.K. Ltd.) and ligated into the unique NotI site in pUTminiTn5km2 resulting in plasmid pJAN25 and maintained in Transformax E. coli strain EC100D pir+ (Cambio U.K.).

Construction of pFEB11

The gene coding for C. jejuni AcrA was amplified by PCR with the ter cassette promoter and chloramphenicol resistance cassette from the plasmid pMH5 as a template using (SEQ ID 17: 5′-TTTTGCGGCCGCTTCTCATGTTTGACAGCTTATC-3′) as forward primer and (SEQ ID 18: 5′-TTTTGCGGCCGCAGCGAGTCAGTGAGCGAGG-3′) reverse primer using the following cycling conditions, 94° C./2 minutes followed by 35 cycles of 94° C. for 30 seconds, 56° C. for 30 seconds and 68° C. for 2 minutes. The product was cut by NotI and cloned into the unique NotI site in pUTminiTn5km2 resulting in plasmid pFEB11 and maintained in Transformax E. coli strain EC100D pir+ (Cambio U.K.).

Construction of pEFNOV19

The gene coding for C. jejuni AcrA was amplified by PCR with the tet cassette promoter and cam resistance cassette from the plasmid pMH5 as a template using (SEQ ID 19: 5′-TTTTGCGATCGCTTCTCATGTTTGACAGCTTATC-3′) as forward primer and (SEQ ID 20: 5′-TTTTAGGCCTAGCGAGTCAGTGAGCGAGG-3′) as reverse primer 94° C./2 minutes followed by 35 cycles of 94° C. for 30 seconds, 56° C. for 30 seconds and 68° C. for 2 minutes. The product was cut by AsisI and StuI (New England Biolabs U.K. Ltd) and cloned in AsisI and StuI digested pJAN25 generating the plasmid pEFNOV19 and maintained in Transformax E. coli strain EC100D pir+ (Cambio U.K.).

Construction of pJCUSA1

We devised a novel transposon cargo loading procedure where the cloning experiments are carried out within a modified version of the commercially available high copy number vector pUC57, this modified plasmid has a chloramphenicol resistance cassette in its backbone from plasmid pMH5 and a Zeocin® resistance cassette flanked by loxP sites allowing for downstream removal of antibiotic marker from the final target strain via the introduction of the CRE enzyme. It has a pMB1 origin of replication and thus can be maintained in any E. coli strain prior to being cut out and transferred along with the Zeocin® resistance cassette using SfiI restriction enzyme digestion and transfer into the pUT delivery vector thus generating a functional transposon. The sequence of this novel construct is shown below (SEQ ID 21):

5′GGCCGCCTAGGCCGCGGCCGCCTACTTCGTATAGCATACATTATACGA AGTTATGTCTGACGCTCAGTGGAACGACGCGTAACTCACGTTAAGGGATT TTGGTCATGATCAGCACGTTGACAATTAATCATCGGCATAGTATATCGGC ATAGTATAATACGACAAGGTGAGGAACTAAAACATGGCCAAGTTGACCAG TGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCC GGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCA GGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA CGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGAC GCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGG GAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCG AGGAGCAGGACTGAATAACTTCGTATAGCATACATTATACGAAGTTATGG CCGCCTAGGCC-3′.

Bacterial Conjugation

To enable transfer of the pglB and acrA transposon cargo into the chromosome of a recipient E. coli strain the plasmids pJAN25, pFEB11 or pEFNOV19 were transferred into the E. coli strain19851 pir⁺ for direct mating. When necessary the donor E. coli strain used was E. coli MFD a diaminopimelic acid (DAP) auxotroph. Growth medium was supplemented with kanamycin 50 μg/ml and ampicillin 100 μg/ml for pJAN25 or pFEB11 whilst chloramphenicol 30 μg/ml and ampicillin 100 μg/ml were added to maintain pEFNOV19. Both donor and recipient bacteria were growth until late exponential phase. Bacterial cells were pelleted by centrifugation, washed 3 times with PBS and mixed together in a ratio of 1:3 recipient to donor and spotted on a dry LB agar plate with no antibiotics for 4-8 hrs. The cells were scraped and suspended in PBS and dilutions plated on LB agar with appropriate selection antibiotics to select for transconjugants. Individual colonies were picked up and screened for loss of the pUT backbone and for the presence of the transposon.

Generation of Unmarked pglB Insertion

The transposon carrying pglB and loxP recombination sites around a Zeocin® resistance cassette was introduced into E. coli. Following selection for Zeocin® resistant colonies, the antibiotic selection marker was removed by introduction via electroporation, the temperature sensitive vector pCRE5 (Reference: Appl Environ Microbiol. 2008 February; 74(4): 1064-1075. Genetic Tools for Select-Agent-Compliant Manipulation of Burkholderia pseudomallei. Kyoung-Hee Choi, Takehiko Mima, Yveth Casart, Drew Rholl, Ayush Kumar, Ifor R. Beacham and Herbert P. Schweizer).

E. coli was cultured at 28° C. in the presence of kanamycin 50 μg/ml, rhamnose was added to induce expression at 0.2% final concentration and the organism subcultured several times to select for colonies that had lost resistance to Zeocin® but maintained resistance to kanmaycin indicating that the bleomycin resistance gene had been flipped out of the chromosome.

This E. coli mutant was then sub-cultured at 42° C. to cure out the pCRE5 plasmid. Screening for colonies that had once again become sensitive to kanamycin confirmed loss of pCRE5 and completed generation of an unmarked inducible copy of pglB on the chromosome of E. coli.

To test glycosylation, this E. coli strain was transformed with the plasmid pACYCpglB::kan (Linton Molecular Microbiology (2005) 55(6), 1695-1703 Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway. Dennis Linton, Nick Dorrell, Paul G. Hitchen, Saba Amber, Andrey V. Karlyshev, Howard R. Morris, Anne Dell, Miguel A. Valvano, Markus Aebi and Brendan W. Wren) and the plasmid pWA2 coding for the HIS tagged acceptor protein AcrA (PNAS vol. 102, pp 3016-3021. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Mario F. Feldman, Michael Wacker, Marcela Hernandez, Paul G. Hitchen, Cristina L. Marolda, Michael Kowarik, Howard R. Morris, Anne Dell, Miguel A. Valvano, and Markus Aebi).

10 ml of LB broth carrying 30 μg/ml of chloramphenicol and 100 μg/ml of ampicillin to select for pACYCpglB::kan were inoculated with a single colony of E. coli strain W3110pglB.

Cultures were incubated at 37° C. with shaking until an optical density 600 nm of 0.6 was reached. IPTG was added to a final concentration of 1 mM and the cultures were incubated for a further 16 hours overnight. Protein was HIS purified and western blots carried out using anti campy glycan and anti HIS antibodies and the images visualised on an Odyssey LI-COR® scanner.

EXAMPLE 1

The E. coli strain W3110 carrying a plasmid coding for a Streptococcus pneumonia capsular polysaccharide encoded on the plasmid pB4-4 was subjected to conjugation with E. coli 19851 pir+ carrying the plasmid pFEB19 coding for an IPTG inducible copy of pglB and a constitutively expressed copy acrA. Previous attempts to make a glycoconjugate using the standard three plasmid system failed presumably due to the combined toxicity of PglB and the metabolic burden of the polysaccharide coding region. The transposon system resulted for the first time in a stable strain. IPTG was added to induce chromosomal expression of pglB and HIS tagged AcrA was purified from the cells using NiNTA binding. FIG. 6 demonstrates how AcrA is shifted in size due to the addition of glycan to the acceptor protein (lane 2). This shift in size does not occur when the glycan is not present (lane 1).

EXAMPLE 2

We have used the transposon pJAN25 carrying an IPTG inducible copy of CjpglB to integrate this gene into the chromosomes of glycoengineering E. coli strains W3110, CLM24, CLM37, Se874, SCM7, SCM6, SCM3.

EXAMPLE 3

We have used the transposon pJAN25 carrying an IPTG inducible copy of CjpglB to integrate this gene into the chromosomes of the following organisms: Vibrio cholerae, Yersinia pseudotuberculosis, Citrobacter freundii, Actinobacillus pleuropneumoniae. In the case of Vibro cholerae and Actinobacillus pleuropneumoniae the standard glycoengineering plasmids used in the tri-plasmid system could not be normally maintained in these organisms.

EXAMPLE 4

pJCUSA1 has been used to generate a transposon with an IPTG inducible copy of CjpglB and a Zeocin® resistance cassette flanked by loxP recombination sites. This was delivered into a recipient E. coli strain by conjugation. This strain can be cured of Zeocin® resistance by the introduction of a CRE recombinase coding plasmid that has a temperature sensitive origin of replication leading to an unmarked E. coli strain capable of generating a glycoconjugate. This is illustrated in FIG. 7. The method is used to generate an unmarked chromosomal insertion of the oligosaccharyltransferase coding gene pglB which is exemplified by glycosylation of an acceptor protein.

EXAMPLE 5

Demonstration of the integration of Campylobacter jejuni PglB into the chromosome of Actinobacillus pleuropneumoniae serotype 8.

E. coli Mu free donor cells (MFD) were transformed with the plasmid pJAN25: Coding for an IPTG inducible copy of CjPglB carried on a pUTminiTn5km2 transposon backbone. This donor strain was conjugated with Actinobacillus pleuropneumoniae serotype 8 and colonies screened for gain of resistance to kanamycin and no resistance to ampicillin indicating that the transposon had left thebackbone and inserted into the chromosome of A. pleuropneumoniae.

To demonstrate that this chromosomally inserted copy of pglB was active, membranes were isolated from the newly constructed mutant of A. pleuropneumoniae after expression of pglB was induced. 16 h A. pleuropneumoniae cultures were diluted 1:25 in fresh media and grown to an OD600 of 0.4-0.6. Expression was induced at this point by addition of IPTG to 1 mM. Incubation was resumed for a further 16 h. Cells were harvested by centrifugation.

Solubilized membranes were prepared by isolating cells expressing CjPglB and by re-suspending the bacterial cell pellet in 2 ml 50 mM Tris-HCl (pH 7.0), 25 mM NaCl and then lysing the pellet using a GE50 ultrasonic processor. Lysates were centrifuged twice at 40 000×g, 4° C. for 20 min to remove unbroken cells. The supernatant was ultracentrifuged at 100 000×g, 4° C. for 1 h, and the resulting total membrane pellet resuspended in 2 ml 50 mM Tris-HCl (pH 7.0), 25 mM NaCl, 1% Triton X-100 per gram of initial wet cell pellet. Samples were incubated at room temperature for 1 h then ultracentrifuged once more at 100 000×g, 4° C. for 1 hour. Supernatants were collected as Triton X-100-solubilised membrane fractions.

The in vitro assay was performed by preparing reaction mixtures that typically contained 3 μl of the fluorescently-labelled peptide FAM-ADQNATA-NH2 (30 μM), 1 μl MnCl2 (150 mM), 4 μl membranes from A. pleuropneumoniae expressing CjPglB and 22 μl membrane preparation from E. coli expressing the C. jejuni heptasaccharide. Samples were incubated at 30° C. with agitation for 16 h. Reactions were stopped by the addition of 10 μl 4×SDS-PAGE gel loading buffer and incubated at 95° C. for 5 min. Samples were loaded on a 4-16% Tricine gel and separated by SDS-PAGE at 30 V for 30 min followed by 200 V for 60 min. Fluorescent peptides were visualized using a Typhoon Trio Imager (FIG. 8). 

The invention claimed is:
 1. A genetically modified bacterial cell, comprising: i) a transcription cassette comprising the nucleic acid molecule of SEQ ID NO: 22; or ii) transcription cassette comprising a nucleic acid molecule with a nucleic acid sequence that is degenerate with respect to the nucleotide sequence of SEQ ID NO: 22 and encodes an oligosaccharyltransferase enzyme comprising the amino acid sequence of SEQ ID NO: 23; and a nucleic acid molecule encoding one or more heterologous polysaccharide biosynthetic genes.
 2. The genetically modified bacterial cell of claim 1, wherein said cell is further transformed with a nucleic acid molecule encoding an acceptor polypeptide for an oligosaccharide or monosaccharide, wherein the acceptor polypeptide includes an amino acid motif which is a substrate for the oligosaccharyltransferase enzyme.
 3. The genetically modified bacterial cell of claim 1, wherein the transcription cassette comprises a regulatable promoter functionally linked to the nucleic acid molecule of SEQ ID NO: 22 or to the nucleic acid sequence that is degenerate with respect to the nucleotide sequence of SEQ ID NO: 22 encoding said oligosaccharyltransferase enzyme.
 4. The genetically modified bacterial cell of claim 3, wherein said regulatable promoter comprises an inducible nucleotide element conferring regulatable expression in response to an inducer.
 5. The genetically modified bacterial cell of claim 3, wherein said regulatable promoter comprises a repressible nucleotide element conferring regulatable expression in response to a repressor.
 6. The genetically modified bacterial cell of claim 1, wherein the transcription cassette comprises a constitutive promoter conferring constitutive expression of the nucleic acid molecule of SEQ ID NO: 22 or the nucleic acid sequence that is degenerate with respect to the nucleotide sequence of SEQ ID NO: 22 encoding said oligosaccharyltransferase enzyme.
 7. The genetically modified bacterial cell of claim 2, wherein said acceptor polypeptide comprises the amino acid motif Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid except proline.
 8. The genetically modified bacterial cell of claim 7, wherein said acceptor polypeptide comprises the amino acid motif: D/E-X—N-X-S/T, wherein X is any amino acid except proline.
 9. The genetically modified bacterial cell of claim 1, wherein said one or more heterologous polysaccharide biosynthetic genes encode capsular polysaccharides or O antigens.
 10. The genetically modified bacterial cell of claim 1, wherein said bacterial cell is a human pathogen.
 11. The genetically modified bacterial cell of claim 2, wherein said bacterial cell is a human pathogen.
 12. The genetically modified bacterial cell of claim 10, wherein said human pathogen is an Neisseria, Moraxella, Escherichia, Salmonella, Shigella, Pseudomonas, Helicobacter, Legionella, Haemophilus, Klebsiella, Enterobacter, Cronobacter or Serratia.
 13. The genetically modified bacterial cell of claim 11, wherein said human pathogen is an Neisseria, Moraxella, Escherichia, Salmonella, Shigella, Pseudomonas, Helicobacter, Legionella, Haemophilus, Klebsiella, Enterobacter, Cronobacter or Serratia.
 14. The genetically modified bacterial cell of claim 1, wherein said bacterial cell is a non-human pathogen.
 15. The genetically modified bacterial cell of claim 2, wherein said bacterial cell is a non-human pathogen.
 16. The genetically modified bacterial cell of claim 14, wherein said non-human pathogen is a Mannheimia spp., Actinobacillus spp., Pasteurella spp., Haemophilus spp. or Edwardsiella spp.
 17. The genetically modified bacterial cell of claim 15, wherein said non-human pathogen is an Mannheimia spp., Actinobacillus spp., Pasteurella spp., Haemophilus spp. or Edwardsiella spp.
 18. The genetically modified bacterial cell of claim 1, wherein said bacterial cell is a zoonotic bacterial species.
 19. The genetically modified bacterial cell of claim 18, wherein said zoonotic bacterial species is a Brucella spp., Campylobacter spp., Vibrio spp., Yersina spp. or Salmonella spp.
 20. A process for producing one or more glycoconjugates, comprising: i) culturing the genetically modified bacterial cell of claim 1 in a cell culture medium; and ii) isolating one or more glycoconjugates from the genetically modified bacterial cell or cell culture medium.
 21. A process for producing one or more glycoconjugates, comprising: i) culturing the genetically modified bacterial cell of claim 2 in a cell culture medium; and ii) isolating one or more glycoconjugates from the genetically modified bacterial cell or cell culture medium.
 22. A transposon, comprising: a transcription cassette adapted for expression in a bacterial host cell, wherein the transcription cassette comprises: i) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22; or ii) a nucleic acid molecule with a sequence that is degenerate with respect to the nucleotide sequence of SEQ ID NO: 22 and encodes an oligosaccharyltransferase enzyme comprising the amino acid sequence of SEQ ID NO: 23; and a nucleic acid molecule encoding a bacterial resistance marker gene.
 23. An isolated nucleic acid molecule, comprising a transcription cassette adapted for expression in a bacterial host cell, wherein the transcription cassette comprises: i) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22; or ii) a nucleic acid molecule with a sequence that is degenerate with respect to the nucleotide sequence of SEQ ID NO: 22 and encodes an oligosaccharyltransferase enzyme comprising the amino acid sequence of SEQ ID NO:23, and a heterologous promoter operably linked to the nucleic acid molecule of i) or ii).
 24. The isolated nucleic acid molecule of claim 23, wherein said transcription cassette further comprises a nucleic acid molecule encoding a bacterial resistance marker gene.
 25. The isolated nucleic acid molecule of claim 24, wherein said bacterial resistance gene confers resistance to an antibiotic.
 26. The genetically modified bacterial cell of claim 16, wherein said non-human pathogen is an Actinobacillus pleuropneumoniae.
 27. The genetically modified bacterial cell of claim 17, wherein said non-human pathogen is an Actinobacillus pleuropneumoniae. 